PROJECT SUMMARY There is an increased interest in peptide medicines in pharmaceutical research and development (R&D) because peptides are recognized as highly selective and efficacious, and at the same time relatively safe and well tolerated. Chemo-enzymatic peptide synthesis (CEPS) using peptide ligases features excellent purity and yield, thus becoming an attractive method to replace traditional chemical synthesis method for synthesizing peptide drugs. However, the development of efficient and versatile peptide ligases lags behind, limiting the advancement of peptide therapeutics. Fundamentally, this is due to the inefficiency of current peptide ligase engineering method that relies on rational protein engineering coupled with low throughput enzymatic characterization. We will overcome this limitation by developing a specialized microfluidic system for high throughput peptide ligase engineering. We will merge modern biochemistry and molecular biology methods with advanced droplet microfluidics to enable high throughput screening of peptide ligase variants. In this project, we will build an enzyme screening platform and demonstrate its capacity on increasing aminolysis to hydrolysis ratio of subtiligase (a well characterized peptide ligase). In Specific Aim 1, we will develop a microfluidic system for ultrahigh-throughput and quantitative analysis of subtiligases. We will develop the microfluidic hardware, processes, and assays to enable the analysis and screening of a large number (over 106) of variants of subtiligase. In Specific Aim 2, we will establish and test subtiligase screening platform. We will design and synthesize subtiligase variant library and we will demonstrate the throughput and sensitivity of our microfluidic system using a mock subtiligase variant library. We will also establish kinetic assays for charactering subtiligase. Achieving these aims will prove that peptide ligase assay with high sensitivity can be incorporated with droplet microfluidic components to enable high throughput engineering of peptide ligase. A proposed phase II project would involve screening of subtiligase libraries to increase aminolysis to hydrolysis ratio of subtiligase and applying our engineering system to engineer other important properties of peptide ligases such as substrate selectivity and racemization activity as well as early production of hardware and disposables.